This invention relates generally to the use of therapeutic agents to regulate immune function. More specifically, this invention relates to the use of inhibitors of the PKA/cAMP pathway to restore T-cell proliferation and to improve immune function impaired in patients infected with Human Immunodeficiency Virus (HIV).
HIV infection leads to immunological disregulation, dysfunction, and abnormalities that result in the development of AIDS. Although less than 1% of lymphocytes are infected with HIV, the vast majority of lymphocytes demonstrate defective function. Drugs, such as AZT, currently approved for treatment of HIV infection, are aimed at inhibiting viral replication rather than restoring defective immune function.
It has been shown that lysates of HIV are capable of impairing proliferation of normal peripheral blood mononuclear cells (PBMC). Pahwa, S., et al., Proc Natl Acad Sci USA (1985) 82:8198-8202, showed that a disrupted HTLV-III viral preparation in relatively dilute concentrations stimulated immunoglobulin secretion by peripheral blood lymphocytes, but at the same dosages was inhibitory for B-cell differentiation responses induced by polyclonal B-cell activators. More recently, Hofmann, B., et al., Cell Immunol (1989) 121:336-348, showed that detergent-disrupted HTLV-IIIB lysate exerted a strong suppressive effect on PHA-stimulated lymphocytes, although the lysate was not directly cytotoxic to lymphocytes and did not contain tumor necrosis factor or lymphotoxin. It was shown that the lysate specifically suppressed the proliferation of a range of hematopoietic cell lines. Thus, it is clear that live virus is not necessary to inhibit the proliferation of PBMC.
In a more recent paper, Hofmann, B., et al., J Immunol (1990) 145:3699-3705, showed that inactivated HIV inhibited the phosphatidyl inositol-4,5-biphosphate and phosphatidic acid-mediated mechanisms of cell activation. This paper showed that treatment of mononuclear cells with inactivated HIV resulted in a decreased inositol phospholipid turnover, leading to a decreased generation of diacyl glycerol (DAG), presumably interfering with phosphokinase C (PKC) activation. As PKC is required for proliferation, presumably diminution of the PKC concentration would result in reduced proliferation, which was also demonstrated in the Hofmann paper. The paper also shows that the expression of receptors for IL-2 and transferrin is decreased.
An alternative mechanism for diminished PKC activity involves stimulation of phosphotyrosine kinases (PTKs), which, in conjunction with the secondary messenger, cyclic AMP, stimulates phosphokinase A activity. Elevated phosphokinase A (PKA) activity is known to inhibit PKC.
As shown hereinbelow, proteins derived from HIV are able to activate this pathway. HIV components induce a signal in T lymphocytes which leads to activation of several protein tyrosine kinases (PTK) in both CD4 and CD8 cells. This is followed by induction of protein kinase A (PKA) and an increase in intracellular cAMP concentrations, which are necessary for the activation of PKA.
Accordingly, the invention is directed to methods to stimulate the proliferation of PBMC so as to ameliorate the effects of AIDS infection. These methods include treating T-lymphocytes by administering medicaments which inhibit HIV-induced increases in cAMP/PKA activity. Increased cAMP/PKA activity results in changes that impair proliferation when cells are stimulated with phytohaemagglutinin (PHA), including lowered membrane protein kinase C (PKC) activity.
One set of agents relevant to such inhibition is directed to adenylate cyclase. A number of inhibitors of adenylate cyclase, the enzyme required for the formation of CAMP, are known, including the Chinese herb extract xe2x80x9cdan-shenxe2x80x9d.
Some of these inhibitors have been used as antivirals. For example, U.S. Pat. No. 4,861,759 discloses compositions containing 2xe2x80x2,3xe2x80x2-dideoxyadenosine, 2xe2x80x2,3xe2x80x2-dideoxyinosine, and dideoxyguanosine and their triphosphates for use in treating retroviral infections.
Nokta et al. (Virology 181:211-217, 1991) demonstrated that HIV infection of MT-4 and other tumor cells was associated with an increase in intracellular levels of cAMP and cGMP. These results are different from those reported hereinbelow in that the target cells were exclusively tumor cell lines, and the elevation in levels of cAMP and cGMP were detected only after very long periodsxe2x80x94i.e., five days after infection. Importantly, the work described in this article has to do with direct viral infection rather than viral lysates; indeed, when a xe2x80x9ccontrolxe2x80x9d using contact with viral protein was tested, there was no detectable increase in cAMP or cGMP levels after five days. Accordingly, Notka concluded that live virus was required to elevate the levels of these cyclic nucleotides.
In an abstract published in connection with the Sixth Annual Conference on Clinical Immunology, Nov. 1-3, 1991, the present inventors disclosed that HIV components induced a signal into T lymphocytes which led to the activation of several PTKs in both CD4 and CD8 T-cells. By use of the specific inhibitor Tyrphostin 25, it was shown that PTK activity was responsible for subsequent induction of PKA, which was, also, accompanied by an increase in intracellular cAMP, necessary for activation of PKA. The inventors herein concluded in this abstract that HIV-induced increased cAMP/PKA activity resulted in changes that impaired proliferation when cells were subsequently stimulated with PHA, including lowering the levels of membrane PKC. A specific inhibitor of PKA (H89) was successful in increasing PKC activity. However, bromo-cAMP and cholera toxoid, reagents which augment intracellular cAMP, resulted in decreased PKC activity.
The present invention concerns methods and compositions for enhancing the function of peripheral blood mononuclear cells (PBMC), thus sustaining the ability of the immune system to provide suitable functionality. This is of particular importance in subjects infected with HIV, since the viral components, not necessarily live viruses, are sufficient to inhibit lymphocyte, especially PBMC, proliferation and impair function. The methods of the invention are directed to protocols that inhibit the PKA/cAMP pathway.